Field of the Invention
This invention relates to expression vectors containing transcription regulatory elements are active in root hair cells in gymnosperms, dicots, and monocots. This invention also relates to genetically altered plants that contain an expression vector containing a heterologous polynucleotide operably linked at the 3′ end and 5′ end to these transcription regulatory elements.
Description of Related Art
Genetically altered plants are being used to solve various agricultural problems, environmental, pest infestation, low yield, etc. One method of generating genetically altered plants, one operably links a promoter with a polynucleotide encoding the gene of interest and introduces the heterologous DNA into a wild-type plant to generate the desired genetically altered plant. Of course one may need to screen the transformed plants to select the genetically altered plant, and the genetically altered plant's progeny, for the desired trait/gene product.
A variety of different types or classes of promoters can be used in genetically altered plants. Promoters can be classified on the basis of characteristics, such as temporal or developmental range, levels of transgene expression, or tissue specificity. For example, a constitutive promoter continuously expresses a gene with minimal regulation. Therefore, promoters referred to as constitutive promoters are capable of transcribing operably linked polynucleotides efficiently and expressing those polynucleotides in multiple tissues.
Numerous promoters, which are active in plant cells, have been described in the literature. Non-exhaustive examples include the nopaline synthase (nos) promoter and octopine synthase (ocs) promoter which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens (also known as Rhizobium radiobacter), and the caulimovirus promoters such as the Cauliflower Mosaic Virus (CaMV) 19S or 35S promoter (U.S. Pat. No. 5,352,605), CaMV 35S promoter with a duplicated enhancer (CaMVE35S, U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938; 5,359,142; and 5,424,200), and the Figwort Mosaic Virus (FMV) 35S promoter (U.S. Pat. No. 5,378,619). These promoters and numerous others have been used in the creation of constructs for transgene expression (expression of heterologous DNA) in plants. Other useful promoters for expression of heterologous DNA are described, for example, in U.S. Pat. Nos. 5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,614,399; 5,633,441; 6,232,526; and 5,633,435.
While previous work has provided a number of promoters useful to direct transcription in genetically altered plants, there is still a great need for novel promoters with beneficial expression characteristics. In particular, there is a need for promoters that are capable of directing expression of heterologous genes or polynucleotides in the root hair cells of genetically altered plants.
Plant technologies which target the root-soil interface or surrounding rhizosphere via genetic engineering require transcription regulatory elements capable of directing accurate and high-level expression of heterologous polynucleotides within root hair cells. Moreover, the use of root hair-specific transcription elements could circumvent adverse effects, such as, but not limited to, potential reductions in crop yield resulting from non-cell type-specific expression of inhibitory gene products.
A plant's root hairs account for a majority of the total surface area of the plant's root systems, and represent the primary sites for nutrient (including mineral) and water uptake, interactions with soil microbes, as well as infection by nitrogen-fixing rhizobia leading to nodulation in legumes. See, e.g., Grierson and Schiefelbein, Root Hairs pp. 1-22 in The Arabidopsis Book, Somerville and Meyerowitz (eds.), American Society of Plant Biologists, Rockville, Md. (2002) (doi/10.1199/tab.0032; www.aspb.org/publications/arabidopsis); and Libault, et al., Trends Plant Sci. 15:641-650 (2010). Thus, numerous biotechnological applications exist for highly active root hair-specific gene promoters, and other polynucleotide sequences influencing steady-state transcript levels within these cells.
A number of studies have involved functional characterization of root hair promoters using promoter:reporter gene fusion constructs (cassettes or expression vectors). See, e.g., Kim, et al., Plant Cell. 18:2958-2970 (2006); Won, et al., Plant Physiol. 150:1459-1473 (2009); and Zhiming, et al., Plant J. February 11. doi:10.1111/j. (2011). However, these studies' goal was the elucidation of regulatory networks involved in root hair transcription, or the physiological role of the associated gene product, rather than identifying highly active promoters for driving heterologous DNA expression.
The root hairs of Sorghum spp. represent a particularly intriguing experimental system, which, to all appearances, serve as high-throughput production “facilities” for allelochemical biosynthesis and rhizosecretion, in addition to the above-mentioned functions (Weston, et al., J. Chem. Ecol. 39:142-153 (2013); Baerson, et al., J. Biol. Chem. 283:3231-3247 (2008)). A prior gene ontology analysis of genes expressed in Sorghum bicolor genotype BTx623 root hair cells revealed that a major proportion of transcriptional activity was associated with “metabolism” (approximately 11.2% of all functions assigned), consistent with previous ultrastructural studies suggesting a high level of metabolic activity for this cell type, likely associated with exudate production and membrane biogenesis (Parker, et al., Plant Cell 12:1961-1974 (2000); Czarnota, et al., Weed Technol. 15:813-825 (2001); Czarnota, et al., Int. J. Plant Sci. 164:861-866 (2003); Baerson, et al. (2008)). Not surprisingly “cellular transport, transport mechanisms, and transport facilitation” was also identified as one of the major functional categories (approximately 7.9% of all functions assigned), given the pivotal role played by root hair cells in soil mineral and organic nutrient uptake (Cutter, The Epidermis in Plant Anatomy pp. 94-106, Clowes & Sons (London, England) (1978); Grierson and Schiefelbein (2002); Libault, et al. (2010)), and the additional specialization required of root hair cells of Sorghum spp. which synthesize and secrete large quantities of the allelochemical sorgoleone into the surrounding rhizosphere (Bertin, et al., Plant Soil 256:67-83 (2003); Weston, et al. (2013)).
As more genetically altered plants are developed in response to diseases and the need to increase yield for food products, a need exists for transcription regulatory elements capable of directing strong root hair-specific transgene expression. This invention is directed at promoters, used with or without specific 3′ flanking regions (terminators), which direct high-level root hair-specific expression of heterologous DNA in both monocotyledonous plants and dicotyledonous plants and the methods of using the same. The regulatory elements described herein deliver recombinant gene products to root hairs at significantly higher levels than is possible using prior art promoters. See, e.g., Kim, et al. (2006); Won, et al. (2009); and Zhiming, et al. (2011).